1. Field of the Invention
The invention relates generally to a method of screening the activity of antioxidant compounds and, more particularly, to a high throughput method for screening the activity of antioxidant compounds at ambient temperatures to more accurately determine antioxidant activity in actual use.
2. Background of Related Art
Antioxidant research is a time-consuming activity when protected samples are evaluated under normal storage conditions. Oxidation at room temperature or below is relatively slow. The signs of emerging rancidity typically only appear after several weeks or even months. For an efficient evaluation of antioxidant systems faster methods of analysis are needed. Many accelerated methods such as Oxidative Stability Instrument (Omnion Inc. Illinois, USA) or OSI, oxygen bomb and Rancimat (Metrohm, CH) use increased temperature to initiate and accelerate the oxidation process. The relation between the rate of any chemical reaction (including oxidation) and temperature can be illustrated using the Arrhenius equation:
                    k        =                  A          ⁢                                          ⁢                      ⅇ                                          -                                  E                  A                                            RT                                                          (        1        )            where k is the rate constant, A is the frequency factor, e is the mathematical constant, EA is the activation energy, and R is the gas constant. The complete term
                    ⅇ                              -                          E              A                                RT                                    (        2        )            counts for the fraction of molecules present in a gas that have energies equal to or in excess of activation energy at a particular temperature. The frequency factor A includes the frequency of collisions and their orientation. This last parameter varies with temperature, although not much. It can be taken as constant across small temperature ranges.
It is possible to calculate what happens to a chemical reaction when the temperature is increased by 10° C., for example from 20 to 30° C. (293 K to 303 K). Because the frequency factor A can be considered constant for this small temperature change, it is only necessary to calculate how equation (2) changes due to the increased temperature. For a typical activation energy of 50 kJ/mol, the following results are obtained:
            For      ⁢                          ⁢      293      ⁢                          ⁢      K      ⁢              :            ⁢                          ⁢              ⅇ                              -            50.000                                8.31            *            293                                =          1.21      ⁢                          ⁢              10                  -          9                                For      ⁢                          ⁢      303      ⁢                          ⁢      K      ⁢              :            ⁢                          ⁢              ⅇ                              -            50.000                                8.31            *            303                                =          2.38      ⁢                          ⁢              10                  -          9                    
These calculations show that the fraction of the molecules able to react has almost doubled by increasing the temperature by 10° C. This causes the rate of reaction to almost double. This is the merit of this rule-of-thumb often used in simple reaction rate work. As for most simple rules it is only an approximation and therefore should be used with great care for the estimation of shelf life. There are a few other important disadvantages to the use of accelerated oxidation at high temperatures:
First of all, there are limitations to the applicability of Arrhenius' law. The rate constant increases as the temperature goes up, but the rate of increase falls off quite rapidly at higher temperatures. This means that there is no linear correlation between tests at higher temperatures and the actual storage temperature. Also the reaction mechanism of the oxidation process typically changes at higher temperature. Consequently the activation energy of the new mechanism probably will be different, and the linear correlation is lost (Frankel, E. N. Stability methods. In Lipid Oxidation; The Oily Press Ltd: Dundee, Scotland; 1998, Vol. 10, pp. 99-114).
Secondly the high temperature of analysis may change the food or feed matrix. Emulsions can break easily, fats will melt, proteins will denature and coagulate (e.g., meat will be cooked), and water may evaporate out of the product. All these transitions can change the matrix considerably and therefore have a dramatic effect on the correlation of the analysis with the shelf life of the original product.
The analysis of more volatile antioxidants is difficult at high temperatures because an unknown amount of antioxidant activity can be lost during the measurement.
Besides increased temperature it is also possible to use metal ions to increase the oxidation rate (Yoshida, Y.; Niki, E. Oxidation of Methyl Linoleate in Aqueous Dispersions Induced by Copper and Iron. Arch. Biochem. Biophys. 1992, 1, 107-114; Yoshida, Y.; Niki, E. Oxidation of Phosphatidylcholine Liposomes in Aqueous Dispersions Induced by Copper and Iron. Bull. Chem. Soc. Jpn. 1992, 1849-1854). Obviously this is not an option for antioxidant research because a high level of metal ions interferes with chelators present in formulations and therefore gives erroneous results.
There is a need, therefore, for new methods for initiating and accelerating oxidation at low temperatures.